CN117143878B - 一种特异性靶向sars-cov-2n蛋白的核酸适配体及其应用 - Google Patents
一种特异性靶向sars-cov-2n蛋白的核酸适配体及其应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，具体涉及一种特异性靶向SARS‑COV‑2N蛋白的核酸适配体及其应用，本发明通过筛选特异性靶向SARS‑COV‑2N蛋白的核酸适配体，降低了SARS‑COV‑2的病毒复制水平，合成成本较抗体制备的成本低，且周期短，化学性质稳定，重现性好；在SARS‑COV‑2感染的诊断和治疗方面具有广阔的应用前景和重要的科学、社会价值，特别是它具有抑制SARS‑COV‑2病毒复制的作用，具备临床治疗新型冠状病毒感染的潜力。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种特异性靶向SARS-COV-2N蛋白的核酸适配体及其应用。
背景技术
新型冠状病毒(SARS-COV-2)高度变异性是疫苗研发、临床防治面临的巨大挑战，其核衣壳蛋白(Nucleocapsid protein，N蛋白)在病毒复制过程中表达量高、功能重要、结构保守，是临床理想的检测和治疗的靶标。
适配体又被称为“合成抗体”、“化学抗体”，其化学本质是一条单链寡核酸分子(ssDNA或RNA)折叠成特定三维结构与靶物质高亲和力和高特异性结合。适配体的获得是通过指数富集配体的系统进化技术(Systematic evolution of ligands by exponentialenrichment，SELEX)的体外筛选过程。核酸适配体具有高亲和力、高特异性、可体外合成、可通过修饰改变其功能及药代动力学特性、无免疫原性、经济等特点。基于上述优势开发的核酸适配体药物可特异性阻断靶标的生物功能，例如作为病毒的感染阻断剂、毒素的中和拮抗剂、细胞因子的抑制剂、阻断转录因子的肿瘤治疗药物等。目前，SARS-COV-2缺乏有效的临床一线治疗药物，主要以免疫预防为主，但因其变异速度快，疫苗免疫效果欠佳，因此，筛选出高特异性、高亲和力识别SARS-COV-2的核酸适配体具有重要的科研和临床价值。
发明内容
为了克服上述技术缺陷的不足，本发明提供一种具有高特异性和高亲和力的特异性靶向SARS-COV-2N蛋白的核酸适配体及在制备SARS-COV-2感染治疗药物、制备样品中SARS-COV-2类病毒颗粒的分离富集试剂及在制备样品中SARS-COV-2检测试剂或试剂盒等多方面的应用。
一方面，本发明提供了一种特异性靶向SARS-COV-2N蛋白的核酸适配体，
所述核酸适配体的序列如SEQ ID NO.1所示：
SEQ ID NO.1：GCT GGA TGT TCA TGC TGG CAAAAG GTG TCA CTC CAT TCC TTAGGGGCA CCG GAA GCATCT CTT TAC TTT GACACATCCAGC。
进一步地，本发明提供一种特异性靶向SARS-COV-2N蛋白的核酸适配体，所述核酸适配体包括在严格条件下与SEQ ID NO.1所示的DNA序列杂交的DNA序列。
进一步地，本发明提供一种特异性靶向SARS-COV-2N蛋白的核酸适配体，包括由SEQ ID NO.1所示的DNA序列转录的RNA序列。
另一方面，本发明提供一种核酸适配体衍生物，由本发明提供的核酸适配体连接、改造、修饰而成；
在一些实施例中，与核酸适配体连接的物质包括荧光素标记、同位素标记、治疗性物质、酶标记、生物素标记的一种或多种；
在一些实施例中，核酸适配体的改造的物质包括由所述的核酸适配体的核苷酸序列的骨架衍生出的硫代磷酸酯骨架或由所述的核酸适配体的核苷酸序列改造成的相应锁核酸或肽核酸。
本发明还提供了核酸适配体、核酸适配体衍生物在制备SARS-COV-2感染治疗药物中的应用。
本发明还提供了核酸适配体、核酸适配体衍生物在制备样品中SARS-COV-2类病毒颗粒的分离富集试剂中的应用。
本发明还提供了核酸适配体、核酸适配体衍生物在制备样品中SARS-COV-2检测试剂或试剂盒中的应用。
有益效果：较之现有技术，本发明通过筛选特异性靶向N蛋白的核酸适配体，降低了SARS-COV-2的病毒复制水平，合成成本较抗体制备的成本低，且周期短，化学性质稳定，重现性好；在SARS-COV-2感染的诊断和治疗方面具有广阔的应用前景和重要的科学、社会价值，特别是它具有抑制SARS-COV-2病毒复制的作用，具备临床治疗新型冠状病毒感染的潜力。
附图说明
图1为核酸适配体的二级结构的生物信息学模拟图；
图2为核酸适配体与SARS-COV-2N蛋白在HEK293T细胞中的共定位情况图；
图3为核酸适配体与SARS-COV-2N蛋白结合的特异性检测图；
图4为核酸适配体与SARS-COV-2N蛋白结合的亲和力检测图；
图5为核酸适配体的抑制SARS-COV-2病毒复制的效果图。
具体实施方式
为使本领域技术人员更好的理解本发明的技术方案，下面结合具体实施方式对本发明作详细说明。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1核酸适配体(N-Apt17)的获取
合成单链DNA文库，其序列如下
GCTGGATGTTCATGCTGGCAAA-N40-TTACTTTGACACATCCAGC；N40：40个随机碱基序列。
原核表达并得到纯化的SARS-COV-2N蛋白，使用SELEX技术筛选到可以识别N蛋白的高亲和力的核酸适配体N-Apt17，其序列为：
SEQ ID NO1：GCT GGA TGT TCA TGC TGG CAA AAG GTG TCA CTC CAT TCC TTAGGG GCA CCG GAA GCA TCT CTT TAC TTT GAC ACA TCC AGC，利用Mfold网络平台分析核酸适配体N-Apt17序列的二级结构，分析出核酸适配体N-Apt17序列的二级结构示意图如图1所示。
实施例2核酸适配体N-Apt17与SARS-COV-2N蛋白特异性结合分析
使用脂质体转染技术将标记绿色荧光基团的N-Apt17(N-Apt17-FAM)转染入表达SARS-COV-2N蛋白(NCOV2，红色荧光蛋白标记)的HEK293T细胞中，通过激光共聚焦技术观察SARS-COV-2N蛋白和N-Apt17在细胞质中的定位情况(见图2)。图2中可以看到，SARS-COV-2N蛋白分布于细胞质中，与N-Apt17存在共定位。白色标尺长度为10μm。
将SARS-COV-2N蛋白的表达基因克隆入pET28a原核表达载体中，在工程化的大肠杆菌表达菌株(BL21)中诱导表达，然后通过镍离子亲和层析技术获得带有HIS6标签的SARS-COV-2N蛋白(N-HIS6)。使用His抗体磁珠(也称anti-HIS磁珠)固定N-HIS6蛋白。将FAM标记的N-Apt17和阴性对照(随机单链核苷酸序列，NC)分别与磁珠固定的N-HIS6蛋白共孵育，然后使用流式细胞术检测N-HIS6蛋白与N-Apt17结合的特异性(见图3)。图3显示SARS-COV-2N蛋白和N-Apt17具有特异性结合能力。
进一步使用微量热涌动技术技术(MicroScale Thermophoresis，MST)测定了SARS-COV-2N蛋白和核酸适配体N-Apt17结合的Kd值(见图4)。具体步骤为：将FAM标记的核酸适配体N-Apt17与倍比稀释的SARS-COV-2N蛋白溶液均匀混合，在微量热泳动仪中检测每个混合浓度的荧光强度，通过荧光强度的值和稀释浓度形成拟合曲线，得出SARS-COV-2N蛋白与核酸适配体N-Apt17结合的Kd值。图4显示，SARS-COV-2N蛋白与核酸适配体N-Apt17的Kd值为0.26±0.04μM，表明核酸适配体N-Apt17与SARS-COV-2N蛋白具备高度亲和力和特异性。
实施例3核酸适配体N-Apt17抑制SARS-COV-2病毒复制分析
使用脂质体转染技术将不同浓度的的N-Apt17(100nM或200nM)转染入VERO-E6细胞，2小时后使用SARS-COV-2感染细胞，12小时后测定细胞培养上清中病毒的滴度，并收集细胞提取RNA定量分析不同实验组细胞中病毒基因组的拷贝差异(见图5)，实验结果显示：N-Apt17降低了细胞培养上清中病毒的滴度(图5A)，N-Apt17明显降低了细胞内病毒基因组的拷贝数(图5B)。
最后需要说明，上述描述仅为本发明的优选实施例，本领域的技术人员在本发明的启示下，在不违背本发明宗旨及权利要求的前提下，可以做出多种类似的表示，这样的变换均落入本发明的保护范围之内。
Claims (5)
1.一种特异性靶向SARS-COV-2N蛋白的核酸适配体，其特征在于：所述核酸适配体的序列如SEQ ID NO.1所示。
2.一种核酸适配体衍生物，其特征在于：由权利要求1所述的核酸适配体连接、修饰而成；
与核酸适配体连接的物质包括荧光素标记、同位素标记、治疗性物质、酶标记、生物素标记的一种或多种；
核酸适配体修饰的物质包括由所述的核酸适配体的核苷酸序列的骨架衍生出的硫代磷酸酯骨架或由所述的核酸适配体的核苷酸序列修饰成的相应锁核酸或肽核酸。
3.权利要求1所述核酸适配体、权利要求2所述的核酸适配体衍生物在制备SARS-COV-2感染治疗药物中的应用。
4.权利要求1所述核酸适配体、权利要求2所述的核酸适配体衍生物在制备样品中SARS-COV-2病毒颗粒的分离富集试剂中的应用。
5.权利要求1所述核酸适配体、权利要求2所述的核酸适配体衍生物在制备样品中SARS-COV-2检测试剂或试剂盒中的应用。
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